Cells respond to certain external stimuli through the production of specific compounds referred to as "second messengers." One family of second messengers is the group of compounds known as diacyl glycerols (DAGs). DAGs are released from cell membrane phospholipids in response to the binding of agonists, such as hormones and growth factors, to cell-surface receptors, which triggers the hydrolysis of phosphatidyl inositol, phosphatidyl choline or phosphatidyl ethanolamine. DAG binds to the regulatory domain of the inactive cytoplasmic enzyme protein kinase C (PK-C), simultaneously increasing its affinity for calcium while causing it to translocate to the cell membrane, where, in the presence of membrane phospholipids, such as phosphatidyl serine, it becomes fully activated. PK-C then catalyzes phosphorylation of serine and threonine residues of enzyme substrates involved in many cellular processes, including growth, cell differentiation, inflammation, nerve function, tumor promotion, and oncogenic expression, by transfer of the .gamma.-phosphate group of ATP.
Given that PK-C represents a central element that transduces signals generated by a broad range of cell signaling pathways that produce DAGs either directly or indirectly and many dominant oncogenes have proven to function, at least in part, through DAG-generating pathways, PK-C has been identified as a potential target enzyme for therapeutic applications, such as anticancer therapy (Gescher et al., Anti-Cancer Drug Design 4: 93-105 (1989)).
Compounds have been developed that bind either to the catalytic site or the DAG binding site of PK-C (see Wang et al., J. Med. Chem. 37(9): 1326-1338 (1994)). UCN-01, staurosporine and Ro-31-8830 are examples of compounds that bind to the catalytic site of PK-C and inhibit PK-C activity. Phorbol-12-myristate-13-acetate and phorbol-12,13-dibutyrate bind to the DAG binding site as activators of PK-C activity. In contrast, bryostatin-1, 12-deoxyphorbol-13-acetate and 12-deoxyphorbol-13-phenylacetate act as partial antagonists of PK-C activity upon binding to the DAG binding site. All of these agents have proven to have therapeutic potential. For example, promising results have been obtained in clinical trials for treatment of melanoma with bryostatin-1. 12-deoxyphorbol-13-acetate has been shown to inhibit mouse skin tumor promotion by 97%. Staurosporine has been shown to be a potent inhibitor of tumor graft proliferation in a mouse model. Ro-31-8830 has been shown to be active orally in a rat model of inflammation and in cultured human T-cells.
Tumor promoters, such as phorbol esters, e.g., 12-O-tetradecanoylphorbol-13-acetate (TPA), and aplysiatoxins, have been shown to activate PK-C by acting as stable, highly potent DAG equivalents. Unlike DAGS, whose presence in the cell membrane is transient, phorbol esters are not metabolized and are, therefore, able to activate PK-C chronically, bypassing the DAG pathway. Chronic constitutive activation of PK-C is associated with resistance of cancerous cells to antitumor drugs.
Compounds that modulate the PK-C pathway by way of the DAG binding site enable selective binding to PK-C, given that the catalytic sites of the thousand or so kinases are structurally homologous and cross-react. The existence of a number of isoforms of PKC and regiospecific expression of PKC mRNAs enable the further selective activation or inhibition of different isoforms of PKC by different agonists or antagonists, respectively.
Unfortunately, ligands currently available for use in targeting the DAG binding site on PK-C, namely the phorbol esters, bryostatins and ingenols, are extremely complex structures with multiple chiral centers, making synthesis and structural modification not only impractical but extremely problematic. Similarly, the indole alkaloids have proven difficult to assess structurally, in terms of homology to the other classes of compounds, so that rational drug design has proven to be elusive. In contrast, DAGs are synthetically accessible but so low in potency as to be unsuitable for use as drugs. However, in view of their structural accessibility, much focus has been placed on DAG analogues in the hopes of obtaining compounds that are not only easily synthesized but sufficiently active to be pharmacologically useful.
In particular, various compounds comprising a conformationally constrained lactone ring system have been synthesized. For example, bis-butyrolactones (Lee et al., Tetrahedron Letters 33(12): 1539-1542 (1992); Lee et al., Tetrahedron Letters 34(27): 4313-4316 (1993); Lee et al., Tetrahedron Letters 34(27): 4317-4320 (1993); Lee et al., SYNLETT 3:206-208 (1994)), .gamma.- and .delta.-lactones (Teng et al., JACS 114(3): 1059-1070 (1992); Lee et al., Biorg. & Med. Chem. 1(2): 119-123 (1993)), including .alpha.-alkyl-.gamma.-lactones (Lee et al., Biorg. & Med. Chem Letters 3(6): 1101-1106 (1993)), .alpha.-alkylidene-.gamma.-lactones (Lee et al., Biorg. & Med. Chem. Letters 3(6): 1107-1110 (1993)), D-galactono-1,4-lactones (Sharma et al., Bioorg. & Med. Chem. Letters 3(10): 1993-1998 (1993)), 2-deoxy-L-ribonolactones (Marquez et al., Bioorg. & Med. Chem. Letters 4(2): 355-360 (1994); Lee et al., Bioorg. & Med. Chem. Letters 4(11): 1369-1374 (1994)), and .epsilon.-lactones (Lee et al., Bioorg. & Med. Chem. Letters 4(4): 543-548 (1994)) were synthesized to function as DAG analogues but binding of these compounds to PK-C was too low to be pharmacologically useful.
There remains a need, therefore, for DAG analogues with pharmacologically useful activity levels which can be synthesized easily and are not rapidly degraded in vivo. Accordingly, it is an object of the present invention to provide such analogues. It is also an object of the present invention to provide pharmaceutical compositions comprising such analogues and methods of using such analogues as PK-C agonists, including partial agonists, and antagonists, including partial antagonists.
These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.